This chapter deals with and reviews evidence that α-defensins secreted by mucosal Paneth cells contribute to innate immunity against bacteria in the small intestine. Growing experimental evidence has led to acceptance of the concept that species produce antimicrobial peptides constitutively or inducibly in response to infection. In mammals, antimicrobial proteins and peptide genes are expressed by varied differentiated cell lineages, including epithelial cells and phagocytes. The α-defensins are major constituents of azurophilic granules in mammalian phagocytic leukocytes, and they are released by a limited number of epithelial cell lineages. The α-defensins from human and rabbit neutrophils achieve bacterial cell killing by distinctive membrane disruptive mechanisms. Acquired immune responses to infectious agents mediated by T cells and B cells are initiated by specific antigens and are selective, but lymphocytic responses may not be rapid enough to contain acute infections by rapidly growing microorganisms or by pathogens that infect a naive host. Absorptive enterocytes, goblet cells, and Paneth cells of the small intestine elaborate lineage-specific gene products or exhibit activities that contribute to enteric host defense. Paneth cell granules are rich in antibiotic proteins and peptides, and release of these granules contributes to mucosal defense. Paneth cells emerge during cytodifferentiation of the fetal small intestinal endoderm from an intervillous epithelium that leads to cryptontogeny. The inflammatory bowel diseases, Crohn’s disease and ulcerative colitis, have provided links between altered mucosal barrier function and the intestinal microflora.

Recognition and cleavage of mouse procryptdins by MMP-7. In A, samples of procryptdins A, B, and C were incubated overnight with (+) or without (–) MMP-7, and samples of those digests were resolved electrophoretically and stained. Electrophoretic mobilities of individual components are noted at left in descending order as follows: MMP-7, matrilysin; PC, purified procryptdins; Crp, MMP-7-activated cryptdin peptides. As described in the text, MMP-7 is the activating metalloproteinase for all mouse Paneth cell α-defensins. Dashes at right denote, in descending order, the position of 28-, 18-, 15.6-, and 7.6-kDa molecular size markers. In B, the consensus cleavage sites disclosed by protein sequencing of MMP-7 digests of procryptdins A to C are noted by asterisks (☆) that interrupt the procryptdin-1 sequence, and the pound character (#) shows the N terminus of procryptdin intermediates purified from mouse small bowel by Putsep et al. (179) that were not evident in the procryptdin A to C digests. Numerals above the primary structure refer to residue positions, with the initiating Met residue in preprocryptdin-1 as residue #1. Reprinted from reference 10 with permission.

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FIGURE 3

Recognition and cleavage of mouse procryptdins by MMP-7. In A, samples of procryptdins A, B, and C were incubated overnight with (+) or without (–) MMP-7, and samples of those digests were resolved electrophoretically and stained. Electrophoretic mobilities of individual components are noted at left in descending order as follows: MMP-7, matrilysin; PC, purified procryptdins; Crp, MMP-7-activated cryptdin peptides. As described in the text, MMP-7 is the activating metalloproteinase for all mouse Paneth cell α-defensins. Dashes at right denote, in descending order, the position of 28-, 18-, 15.6-, and 7.6-kDa molecular size markers. In B, the consensus cleavage sites disclosed by protein sequencing of MMP-7 digests of procryptdins A to C are noted by asterisks (☆) that interrupt the procryptdin-1 sequence, and the pound character (#) shows the N terminus of procryptdin intermediates purified from mouse small bowel by Putsep et al. (179) that were not evident in the procryptdin A to C digests. Numerals above the primary structure refer to residue positions, with the initiating Met residue in preprocryptdin-1 as residue #1. Reprinted from reference 10 with permission.

Release of microbicidal peptide activity by crypt Paneth cells in response to live bacteria. Crypts (solid circles) or villi (shaded diamonds) were isolated from adult mice and incubated in numbers shown with 103 of S. enterica serovar Typhimurium CFU for 1 h at 37°C in 50 µl of isotonic buffer. Viable CFU were quantitated by plating the entire mixtures on semisolid nutrient media overnight. Data points show surviving bacteria from individual replicate mixtures. Open circles represent surviving bacteria after incubation of 103 CFU for 1 h at 37°C in 50 µl of isotonic buffer in the absence of crypts or villi. As noted in the text, bacteria exposed to crypts die in response to Paneth cell secretions (11), but equivalent exposure to villus epithelium does not affect S. enterica serovar Typhimurium viability. Reprinted from reference 11 with permission.

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FIGURE 4

Release of microbicidal peptide activity by crypt Paneth cells in response to live bacteria. Crypts (solid circles) or villi (shaded diamonds) were isolated from adult mice and incubated in numbers shown with 103 of S. enterica serovar Typhimurium CFU for 1 h at 37°C in 50 µl of isotonic buffer. Viable CFU were quantitated by plating the entire mixtures on semisolid nutrient media overnight. Data points show surviving bacteria from individual replicate mixtures. Open circles represent surviving bacteria after incubation of 103 CFU for 1 h at 37°C in 50 µl of isotonic buffer in the absence of crypts or villi. As noted in the text, bacteria exposed to crypts die in response to Paneth cell secretions (11), but equivalent exposure to villus epithelium does not affect S. enterica serovar Typhimurium viability. Reprinted from reference 11 with permission.

Role of mIKCa1 in Paneth cell secretion. Exposure of Paneth cells to pharmacologic agents (197) or bacterial antigens results in an initial increase in cytosolic Ca2+ ([Ca2+i]) by mobilization of intracellular stores. mIKCa1 channels in the Paneth cell membrane would be predicted to open as cytosolic [Ca2+i] approaches 300 nM, providing the counterbalancing K_ efflux necessary to sustain Ca2+ entry from the external milieu (12). As noted in detail in the text, blockade of mIKCa1 would depolarize the membrane and attenuate the calcium-signaling response required to generate a complete Paneth cell secretory response. Electron micrograph was generously provided by Susan J. Hagen, Beth Israel Deaconess Medical Center, Boston, Mass.

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FIGURE 5

Role of mIKCa1 in Paneth cell secretion. Exposure of Paneth cells to pharmacologic agents (197) or bacterial antigens results in an initial increase in cytosolic Ca2+ ([Ca2+i]) by mobilization of intracellular stores. mIKCa1 channels in the Paneth cell membrane would be predicted to open as cytosolic [Ca2+i] approaches 300 nM, providing the counterbalancing K_ efflux necessary to sustain Ca2+ entry from the external milieu (12). As noted in detail in the text, blockade of mIKCa1 would depolarize the membrane and attenuate the calcium-signaling response required to generate a complete Paneth cell secretory response. Electron micrograph was generously provided by Susan J. Hagen, Beth Israel Deaconess Medical Center, Boston, Mass.

Dense granules in mouse small intestinal intermediate granulomucous cells during T. spiralis infection. (A) This transmission electron micrograph shows evidence of extensive Paneth cell degranulation into the crypt lumen (L). Cells at the top of the field display large electron dense granules within electron-lucent mucus-containing granules, the hallmark of intermediate granulomucous cells. (B) Two representative intermediate cells that were detected above the crypt villus junction. Such cells are immunopositive for mouse Paneth cell α-defensins and are not found after clearance of the infection (101). Reprinted from reference 101, with permission.

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FIGURE 6

Dense granules in mouse small intestinal intermediate granulomucous cells during T. spiralis infection. (A) This transmission electron micrograph shows evidence of extensive Paneth cell degranulation into the crypt lumen (L). Cells at the top of the field display large electron dense granules within electron-lucent mucus-containing granules, the hallmark of intermediate granulomucous cells. (B) Two representative intermediate cells that were detected above the crypt villus junction. Such cells are immunopositive for mouse Paneth cell α-defensins and are not found after clearance of the infection (101). Reprinted from reference 101, with permission.

112.Lasserre, C.,, C.Colnot,, C.Brechot,, and F.Poirier. 1999. HIP/PAP gene, encoding a Ctype lectin overexpressed in primary liver cancer, is expressed in nervous system as well as in intestine and pancreas of the postimplantation mouse embryo. Am. J. Pathol.154:1601–1610.